Antibiotic and production thereof



Nov. 3, 1964 s. PlNNERT ETAL 3,155,581 ANTIBIOTIC AND PRODUCTION THEREOF Filed Feb. 16, 1962 /V/e rmer'f 6 Leon [V1 2 e7 N ean Freud 7207227119 Inventors m r Attorney United States Patent 3,155,581 ANTEBIQTKC AND PRODUCTION THEREGF Sylvie Pinnert, Leon Ninet, and Jean Preudhomme, Paris, France, assignors to Rhone-=Poulenc S.A., Paris, France, a French body corporate Filed Feb. 16, 1962, Ser. No. 173,770 Claims priority, application France Feb. 24, 1961 6 Claims. (Cl. 167-65) The present invention relates to a new antibiotic, designated hereinafter by the number 9671 RP. This new product has important antibacterial activity against Grampositive organisms and more especially staphylococci and streptococci.

This new antibiotic is produced by the culture under artificial conditions of a micro-organism, hereinafter identified more completely, belonging to the genus Streptomyces and designated Streptomyes 40037 or Streptomyces actuosus. A specimen of this micro-organism has been deposited in the United States Department of Agriculture, North Regional Research Laboratory at Peoria, Illinois, United States of America, and is identified by the Culture Collection Number NRRL 2954.

According to a first feature of the present invention, therefore, there is provided an antibiotic, herein designated 9671 RP, which is a yellow substance, crystallising as fine needles, melting point 310320 C. with decomposition on a Maquenne block and having an optical rotation [on] =+38 (C.=l, pyridine), analysing with the elementary composition C, 49.6%; H, 4.0%; O, 16.7%; N, 14.4%; S, 15.7515.80%, having an ultra-violet spectrum in solution in water containing dimethylformamide exhibiting two absorption maxima at exhibiting the following principal infra-red absorption bands, shown in Table I:

3350 strong 3125 shoulder 2930 shoulder 1745 medium 1655 very strong 1532 very strong 1484 strong 1425 medium 1385 weak 1342 medium 843 medium 1308 medium 822 weak 1235 medium 790 medium 1210 medium 752 strong 1 168 medium the said antibiotic being soluble in chloroform, dioxane, pyridine, dimethylformamide and dimethylsulphoxide, slightly soluble in methanol, ethanol, ethyl acetate and benzene and insoluble in water and petroleum ether.

Referring to the foregoing data, the ultra-violet absorption spectrum is conveniently determined using a solution of ug/cm. of the antibiotic in water containing 1% of dimethylformamide, and the infra-red absorption spectrum is conveniently determined using tablets containing the antibiotic and potassium bromide.

The infra-red absorption spectrum of the antibiotic is shown in the accompanying drawing, where the abscissae represent both the weavelengths expressed in microns (lower scale) and the wave numbers in cm.- (upper scale) and the ordinates represent percentage transmiss1on.

The antibiotic 9671 RP gives negative results in the following reactions: biuret reaction, Sakaguchi reaction, ninhydrin, reaction, Millon reaction, Folin-Denis reacice A tion, ferric chloride reaction, Molisch reaction, Fehlings solution reaction, Tollens reaction, Ehrlich reaction, ferric maltol reaction and Zimmermann reaction. It gives a positive result to the ninhydrin reaction after hydrolysis, thus demonstrating the existence of a polypeptide chain.

The antibiotic 9671 RP may be identified by paper chromatography. The antibiotic has been chromatographed on Arches No. 302 paper impregnated with M/ 15 phosphate butter at pH 7, descending development being carried out by means of difierent solvents or mixtures of solvents. The chromatograms are developed by bioautography on nutritive agar plates seeded with S. aureus or B, subtilis. The Rf values obtained are given in Table II:

The bacteriostatic activity'of 9671 RP against organisms has been determined by a dilution method currently used for this purpose. The lowest concentration of substance has been determined for each organism which prevents all visible development in an appropriate nutritive medium. The results obtained in the various determinations have been collected in Table III below, wherein the minimal bacteriostatic concentrations are expressed in micrograms of substance per cm. of experimental medium.

TABLE III Minimal Bacteriostatic Test Microorganism Concentration, pig/0111.

Staphylococcus aureus strain 209 P-ATCC 6538 P. 0.0009 Staphylococcus aurcus strain 133 (Pasteur Institute)- 0.0019 Staphylococcus aurcus strain B (streptomycin and pen cillin resistant). 0. 003 Staphylococcus aurcus strain I-Ib (tetracycline and penicillin resistant) 0. 003 Staphylococcus aurcus strain Launoy 1 (tetracycline,

streptomycin, chloramphcnicol and penicillin resistant). 0. 0038 Staphylococcus aurcus strain Launoy 2 (tetracycline,

streptomycin and penicillin resistant) 0.0021 Staphylococcus aureus strain Bcauj on 3 (tetracycline,

streptomycin, chloramphenicol and penicillin resistant) 0. 003 Staphylococcus aurcus strain 2700Rstreptothricin resistant 0.0025 Staphylococcus uureus strain 1142Rcongocidin resistant. 0. 0005 Staphylococcus aurcus strain 3486Rspiramycin resistant. 0. 0000 Staphylococcus aureus carbomycin resistant strain 0.0005 Staphylococcus aurcus erythromycin resistant strain. 0, 0033 Staphylococcus cureus chloramphenicol resistant stra 0. 0012 Staphylococcus aurcus novobiocin resistant strain. 0. 001 Staphylococcus au'reus actinomycin resistant strain 0.0012 Micrococcus citreusATOO 8411 0. 0038 Micrococouslysodcikticus-ATCC 4098 0.003 Gajfkya telraycua (Fae. Phie) 0. 0042 Sorclna lulcaATCC 9341. 0. 0011 Sorcina alba (Fae. Phie)... 0.0019 Streptococcus fcccalis (Thiercelm Fae. Phie ontcrococcus) 0. 0106 Streptococcus faccalisATOO 9790 0.0007 Streptococcus oirlrlcns (Institut Pasteur) 0. 0065 Streptococcus pyoyeues hcmolytlcus (Dig. 7 Institut; Pasteur strain) 0. 00028 Diplococcus pncumonioe (Til strain, Institnt Pastenr).-.. 0.00015 Neisscria catarrhalz's (Fac. Phie) 0.0017 Lactobactllus casci-ATCC 7409.. 0. 0007 Bacillus subti ATOO 66 0.003 Bacillus suhtilis(strain Z0 5 A, Fae. Ph1e) 0.01 Bacillus sublilis-(strain 3 R 9075, Merck)-A'l CC 9524.. 0.13 Bacillus ccreus-ATCC 6630 0. 0071 Bacillus bracts-ATCQ 2185. 138 Bacillus mycoidcs 0. 0043' .Mycobacterium smcymalis-ATO C v607 8 Mycobaclcriu'm smcymatls-ATOC 607 NR (neornycin' resistant strain) 5.07 Lfycobactcrium smcy-molis-ATCC'607 SR (streptomycin resistant strain) 138 TABLE IKE-Continued sons nutritive medium or any other appropriate medium. After incubation at 26 C. for several days, the colonies im of micro-organisms which it is desired to isolate are T Bacteriosmtic transplanted to agar slopes to produce more abundant est Micro-organism Concona i cultures. No description of a species having culture i s-l characteristics and biochemical properties coinciding with M L M (I tb t 1 d L 0 17 those of S. actuosus can be found in the classification of i i 2. 3 3 9 You the genus Streptomyces in Bergeys Manual of Deterizi minative Bacteriology, 7th Edition (1957). For this states:anaesthetistsass?t???1;.. is; w this Organism may be congested to be a new Salmonella puratyphiA (Lacnssc, Institut Pasteur) 133 species. The name Streptomyces actuosils has been tarriatsiiiiiet ffti teiiei'iiiiiieaefi55 138 given to it of its great activity and the following Bacillus lactis aerogmws (Fae. Phic)- .1 38 are its properties: i s f liii h i o i 3 0015 M 1 f zclt ti gtlgfisfl flc. Pliio) 15 1 lcroscopw orp z g g Cuitures on thin plates of Bennett s medium examined s erm i o t a e s g g g underthe microscope show the format on of branched Bifl dlfl bi michise ii tz'ca (CNhS7-{Vclcome Institt it) l38 mycchal filamsnts as Well as Spore chfuns charactensnc asleurclla vniiltocida a. 125,1nstitut Pasteur) 0.0024 of the genus Streptomyces. The mycelial filaments carry very numerous spore chains. The spore-bearing filaments These results taken together show that 9671 RP is a sinuous and of varying lengths The spores are oval principally active against Gram-positive bacteria. In and abOut in breadth and about in lenglh- The addition, 9671 RP is active against staphylococcal strains mycell'al filaments are more Slender than the p rendered resistant to one or more of the following anti 3 filaments and are about t0 in breadthbiotics: enicillin, streptomycin, tetracycline, choram- I phenicoh erythromycin, novobiocin, actinomycin, stepto- Genaml Chamctensncs thricin, congocidin, spiramycin and carbomycin. On those media described as synthetic S. actuosus 9671 RP is inactive against the following yeast-iike or presents in general a colourless to pale orange vegetative filamentary fungi: Pestalotia palmarum, Sacclzaromyces mycelium and produces no soluble pigment or weak pastorianus, Stemphylium radicinum, Aspergillus niger, ochre or brown pigments. On those media described Candida albicans, Botrytis cinei'ca, Fusarz'um oaysporzmz as organic it presents a vegetative myceliuin the colour d pe i illiu chrysogenum, of which ranges from yellow-brown to dark brown and I dditi it h b d o t t d in th laboratory soluble pigments of a brown which is more or less intense that 9671 RP is particularly active against staphylococcal according to the medium and the age of the culture. infections of the mouse when administered in situ. The Aerial mycelia do not appear on all media. When they toxicity of the antibiotic has been studied principally in are Produced y are always While at the Commencement the mouse. The maximal tolerated dose, i.e., the maxiof their development and then become medium grey in mal dose for which no deaths are recorded amongst the colour. animals (LD has been determined by sub-cutaneous, F of Isolated Colonies oral and intra-peritoneal administration of the product, 40 with the following results: Monospore cultures in Petri dishes on Bennetts mediurn take the form of round colonies without ragged Sub-cutaneous administration, LD above or equal to 1 edges. These colonies are somewhat convex and may possess a central button. The vegetative mycelium forms Oral p aiaove or equal to numerous ray-like folds. It is pale orange in colour as lntm'pentoneal admmlstratloni LD0=0'5 is its underside. The aerial mycelium appears rather Of low toxicity in the mouse and well tolerated in the early. At first white, it then changes with the degree rabbit 011 application to mucous membrane, this product of development to become medium y in colour wh is a therapeutic agent of choice for the local treatment it is mature- It forms 11 light brown Pigment in agarof infections caused by Gram-positive cocci in animals. In particular, it is very efiective in the treatment of the Culture Chm actcrlstlcs and Biochemical Properties mastitis of the cow. It may be also used as a food ad- The culture characteristics and biochemical properties ditive of Streptomyces actuosus have been determined on the As noted above, the organism which produces the N nutritive agars and nutritive broths customarily used to antibiotic 9671 RP belongs to the genus Streptomyces identify strains of Streptomyces. The observations made and is designated by the Culture Collection Number are recorded in Table IV below. They relate to cultures NRRL 2954. It is herein referred to as S. actuosus. incubated at 26 C. for 27 days. The majority of the This organism was isolated from a soil fragment taken culture media employed were prepared according to the near Corrientes in Argentina. The method of isolation formulae contained in The Actinomycetes by S. A. was as follows: the soil sample was suspended in sterile Vlaksman, pp. 193-197, Chronica Botanica Company, distilled water and the suspension then diluted to dif- -Waltham (Mass), United States of America, 1950. ferent concentrations. A small volume of each dilution Where this is the case, they are indicated by the number was spread on the surface of Petri dishes containing Emergiven to them in The Actinomycetes.

TABLE IV Culture Medium Degree of Vegetative Myceliurn Aerial Mycelium Soluble Pigment Biochemical Properties Development Bennett's Agar (Reina). Good Lightly folded, slightly Abundant;mediun1grey Brown and of medium etllorescent. intensity. Glyccrin-Asparagine Moderatecolourless Slight, white growing to Weak and or ochre gar (3 medium grey. colour. Synthetic Agar (Starchr d0 Colourlcss,slightlycfilo Slight, initiallywhitothon Weak and of yellow- Mincrol Salts) (Bet. J). rosccnt. becoming medium grey. ochre colour. Maltose-Tryptone Agar Vigorous Thick and strongly pig- White, chalky appearance Intense and brown (rel. B). Inented (black);f0lded. becoming mcdiunigrcy.

TABLE IV-Continued Culture Medium Vegetative Mycelium Aerial Mycelium Soluble Pigment Biochemical Properties Synthetic Agar (Oza- Good Oolourless, slightly efiio- Abundant, begins to Rather abundant and peks Medium) (1). reseent. become grey very shortly ochre.

after its appearance but remains pale. Synthetic Agar with do do White, powdery, becoin- Rather abundant and Glucose (ref. ing very pale grey. ochre. Emcrsons Agar (23) do Folded and brilliant Greyish white Clear brown.

clear brown.

Glucose-Asparagine Moderate Coloui'less Clear grey with several None gar (2). small white bunches.

Nutritive Agar (5) Weak Math ochre, brown, Absent Ochre, brown,yellow-.

ye 0W.

Glucose Agar (7) Good Smooth, brilliant At first greyish white, None Formation of crystals in chalky appearance, bethe agar. coming pale grey very slightly rose-tinted but with clear and dark zones, the clear zones predominating.

Agar with Starch (10). .0 Moderate. Colourless or pale White, becoming medium Rose ochre orange. grey.

Agar with Calcium Colourless do None Solubilisation oi calcium Mal-ate (ref. D). malate. Agar with Tyrosine Clear brown Absent Brown No solubilisation of the (ref. E). tyrosine. Pu e lat e (Ref. Flakes on the surface of -do Yellow brown Liquefaction or pg of the F). thegelatine. depth in 12 days; of of the depth in 27 days. Potato (27) Vigorous Folded White at the edges; medi- Yellow exudate, 111 um grey at the centre. droplets on the surface of aerial niycelia; brown soluble pigmerit.

Starch Solution (l9) Moderate"--. Granular cloudiness at Absent None Hydrolysis of the starch the surface; flakes at is not complete in 12 the bottom, for the days (reactiontoiodine most part clinging to iodide solution); after the walls of the tube. 27 days only dextrins remain.

Czapeks Broth (l8) do Flakes at the bottom of do Slight and yellow, more the tube. intense towards the surface than at the bottom. Czapoks Broth with l() do do Glucose (ref. G). Nitrated Nutritive Moderate Granular cloudiness at de None Positive reaction for ni- Broth (ref. H). the surface and small trites.

colonies at the bottom adhering to walls. I skimmed Milk (ref. K) Good Cloudy md dark brown Traces, white At first dark brown near On the 12th day no coag- [ing the surface, clearer at ulation, pH 6.8. On the bottom. The 27th day peptonisation brown pigment then is complete, pH 7.2. spreads throughout the entire medium.

References to or constitutions of media not described in Utilisation of Various Hydrocarbon Substances The Actinomycetes A. K. L. Jones, J. Bacteriology, 57, 142 (1949).

B. A. H. Williams, E. McCoy, App. Microbiology, 1,

C. Obtained by replacing the sucrose in the formula given by Waksman for Czapcks synthetic sucrose agar (l) by glucose (3%).

D. Calcium malate 1%; Nl-l Cl 0.05%; K Hl O 0.05%; agar 2%.

E. Peptonc 0.5%; meat extract 0.3%; tyrosine 0.5%;

agar 2%.

F. Plain gclatine prepared according to the instructions in the Manual of Methods for Pure Culture Study of Bacteria of the Society of American Bactcriologists 50-18)- G. Obtained by replacing the sucrose in the formula given by Waksman for Czapeks sucrose broth (18) by glucose (3%).

H. Following the formula given in the Manual of Methods for Pure Culture Study of Bacteria of the Society of American Bacteriologists (II J. Grundy et coil.: Antibiotics and Chemotherapy, 1, V

309, (1951). K. Prepared from Gayelord Hauser powder according to instructions. given.

skimmed-milk (According to the method of Pridham, T. G. and Gottlieb, D., J. Bacteriology, 56, 167 [1948]) The cultures are carried out on agar slopes. incubation is at 26 C. Among the substances inducing a rapid growth, complete with the formation of aerial my'celia and spores in 10 days are: glucose, maltose, lactose, sucrose, starch, dextrin, xylosc, rafinose, ribose, inulin, fructose, marinosc, arabinose, rhamnose, galactose, mannitol, inositol, meso-inositol, glycerol, sodium acetate, sodium citrate and succinic acid.

Sorbose, sorbitol, erythritol, adonitol and dulcitol do not allow development or allow only an extremely restricted development. It can thus be said that these substances are not utilised.

Comparison of S. Actuosus and Two Strains of Streptomyces Described in Bergeys Manual of Determinativc Bacteriology These two strains, Streptomyces fasciculus (originally described by Krassi1nikov) and Streptomyces camo-sus (described by Millard and Burr), are those which most closely approach the strain here referred to as Strepto myces ocluosus.

.The differences in culture characteristics and biochemical properties are listed in Table V.

TABLE V S.fasciculus S. camosus S. aciuosus Aerial myeelium little developed,

grey. Medium liquefaction Cylindrical Vegetative myeclium pale smolky grey-exudate colourless in droplets over entire surface.

Vegetative mycclium pale olive grey.

sinuous.

Vegetative mycelium colourless; no

exudate.

Vegetative mycelium colourless.

No aerial mycelium.

Gelating (F) Rapid liquefaction Liquefaction slow and incomplete. Milk (19---. Rap id coagulation and peptoni- Coagulation followed by liquelae- No coagulation; pcptonisation slow.

so ion. tion. Starch (l9) Rapid hydrolysis Hydrolysis Hydrolysis slow and incomplete I (stops at dextrin stage). Nitrate (H) Weak reduction to nitrite Reduction Good reduction.

According to a further feature of the present invention, the antibiotic 9671 RP defined above is prepared by culturing S. actuosus or a mutant thereof in a nutritive medium and separating the antibiotic 9671 RP thus formed.

The culture of S. actuosus may be carried out by any method of surface or submerged aerobic culture but the latter is preferred for reasons of yield. For this purpose the different types of apparatus which are now currently utilised in the fermentation industries may be used.

In particular, the following route can be adopted for the conduct of the operations:

S. acluosus-stock i Culture on agar Culture in agitated flask Inoculum culture in fermentation vessel Production culture in fermentation vessel The fermentation medium should contain essentially an assimilable source of carbon and an assimilable source of nitrogen, mineral substances and, optionally, growth promoting factors. All of these elements may be in the form of clearly defined substances or complex mixtures such as are found in biological products of diverse origins.

Suitable assimilable sources of carbon are provided by carbohydrates, such as glucose, sucrose, lactose, dextrins, starch and molasses or other hydrocarbon substances, such as the sugar alcohol, glycerol and mannitol, or organic acids such as lactic, citric and tartaric acids. Animal or vegetable oils such as lard or soya-bean oil may, advantageously, replace these different hydrocarbon sources or be added to them.

Suitable assimilable sources of nitrogen are extremely varied. They may be very simple chemical substances such as nitrates, mineral and organic ammonium salts, urea and aminoacids. They may also take the form of complex substances containing nitrogen principally in protein-like form; suitable such materials being, for example, casein, lactalbumin and gluten and their hydrolysates, soya-bean, peanut and fish meals, meat extracts, yeast, distillers solubles and corn-steep.

Among the added mineral substances, certain may have a bufiiering or neutralizing effect, such as alkali-metal or alkaline earth metal phosphates or calcium or magnesium carbonates.

Other elements contribute to the ionic equilibrium necessary for the development of S. actuosus and the elaboration of the antibiotic, such as alkali-metal and alkaline earth metal chlorides and sulphates. Finally, certain elements act more especially as activators of the metabolic reactions of S. actuosus; examples are salt of zinc, cobalt, iron, copper and manganese.

The pH of the fermentation medium at the commencement of the culture is preferably between 6.0 and 7.5. The optimum temperature for the fermentation is 2627 C., but satisfactory production is obtained at temperatures between 23 and 35 C. The aeration of the fermentation may be varied between fairly wide limits. it has, nevertheless, been found that an aeration rate of 0.5 to 2 litres of air per litre of broth per minute is particularly suitable. The maximum yield of the antibiotic is obtained after 3 to 5 days of culture, the time depending essentially on the medium used.

Thus, the general conditions for the culture of S. actuosus for the production of the antibiotic may be varied to a fairly wide degree and adapted to each particular circumstance.

The antibiotic 9671 RP can be isolated from fermentation cultures by various methods. The fermentation culture may be filtered at a pH between 6 and 9 but, in this case, a major part of the active material remains in the filter-cake which must also be treated to extract the product. It is, therefore, preferable to carry out the filtration at a pH range of 1 to 6. In these circumstances the active material remains in the filter-cake from which it can be extracted with a solvent selected from the aliphatic alcohols, such as methanol, ethanol, the propanols or the butanols, ketones such as acetone or methylisobutyl ketone, and esters such as ethyl acetate. It is also possible to extract the fermentation culture with a waterimmiscible organic solvent from the group of aliphatic alcohols containing 4 or 5 carbon atoms, ketones and esters. In this case, the active material passes into the organic phase which is separated from the aqueous phase by filtration or centrifuging.

The crude product can be isolated from the above organic solutions by concentration of the organic solution to a small volume. The crude antibiotic precipitates on cooling or the addition of a poor solvent for 9671 RP such as petroleum ether or cyclohexane. When the antibiotic occurs in the filtrate of the culture, this solution is extracted with a water-immiscible solvent such as an aliphatic alcohol containing 4 or 5 carbon atoms, a ketone such as methylisobutyl ketone, an ester such as ethyl acetate or amyl acetate or a chlorinated solvent such as chloroform or dichlorethane. The procedure given above is then followed, i.e., concentration to a small volume and precipitation.

The antibiotic 9671 RP can then be crystallised by dissolving the precipitate obtained above in acetic acid with gentle heating and then cooling. When the crude product is too impure to be directly crystallised, or a very pure product is required, it is advantageous to submit the crude antibiotic to a two-stage purification. The first stage consists of washing the crude antibiotic with an organic solvent in which it is insoluble or only slightly soluble, for example methanol, ethanol, ben- Zene or petroleum ether, filtering and drying. The second stage consists of chromatographing the antibiotic which has already been partially purified by the washing described above. Chromatography can be carried out using a column of alumina through which a solution of the product, in a suitable solvent, is passed. Suitable solvents are, for example, chlorinated solvents such as chloroform or dichlorethane or mixtures of one of these chlorinated solvents with a solvent in which the antibiotic is poorly soluble such as, for example, methanol or ethanol. The product is eluted with the same solvent. The eluate is concentrated and the antibiotic precipitated by the addition of a poor solvent as described above.

The products which have been thus purified are dissolved with gentle heating in aqueous or glacial acetic acid. After filtration of the solution and the addition of water, crystallisation is carried out by cooling with slow agitation.

The various methods described above may be applied successively in a varied sequence or repeated several times, according to the requirements of manufacture, in order to obtain the antibiotic 9671 RP in a suitable form for its desired use.

The present invention further includes within its scope pharmaceutical compositions which comprise the antibiotic 9671 RP together with a pharmaceutical carrier. In clinical practice the compounds of the present invention will normally be administered topically.

The following examples will serve to illustrate the invention. In these examples activity is determined throughout by a turbidimetric method using Staphylococcus aureus 209 P as the sensitive organism and comparing With a pure crystalline specimen of the product as standard. This activity is therefore expressed in micrograms g) of standard crystalline product per mg. for solid products and in ,ug. of standard crystalline product per cm. for solutions.

EXAMPLE I A fermentation vessel (75 litres) is charged with:

Corn-steep (50% dried extract) g 800 Sucrose g 1200 Ammonium sulphate g 80 Tap Water litres 35 The pH is adjusted to about 5.2 by the addition of sodium hydroxide solution (d=1.33; cc.). Calcium carbonate (300 g.) is then added. The medium is sterilised at 122 C. for 40 minutes by the passage of steam and after sterilisation, and cooling to 27 C., the final volume of the medium is 40 litres and the pH 7.15. The medium is then seeded with 250 cc. of a culture of S. actuosus in an agitated Erlenmeyer flask.

The culture in the fermentation vessel is aerated with sterile air at a rate of 3 m. /hour and agitated with a helix turning at 400 rpm. The temperature is kept at 27 C. The pH of the medium remains at its initial value (7.15) for 4 hours and then falls slowly to reach 6.85. The development of the organism corresponds to this lowering of the pH and it is satisfactory to seed the production culture 28 hours after the initial seeding.

The production culture is carried out in a fermentation vessel (350 litres) charged with the following materials:

Soya flour kg 8 Distillers solubles kg 1 Glucose (hydrated) kg 1 Soya oil litres 4 Calcium carbonate kg 2 Sodium chloride kg 2 Water litres 175 The pH of the medium before sterilisation is 6.95. The medium is sterilised at 122C. for 40 mintues by the passage of steam. After sterilisation, and cooling to 27 C., the final volume of the medium is 200 litres and the pH 7.20. The medium is then seeded with 20 litres of the preceding culture from the 75 litre fermentation vessel. The medium is agitated with a turbine turning at 205 rlptm, aerated with 10 m. /hour of sterile air and the temperature maintained at 27 C. The pH falls slowly for 24 hours after which it rises again. The production of the antibiotic commences with this rise in the pH. The completion of the fermentation is not marked by any alteration in the curve of the rise of pH. It occurs at the end of about hours and the activity of the medium is then 295 ug/CC.

.EXAMPLE II A fermentation culture (190 litres; titre 295 g/cc. as

in Example I) is placed in a container fitted with an agitator. The culture is stirred for 1 hour with butanol litres), the pH being adjusted to 5 with dilute hydrochloric acid. A filtration adjuvant (19 kg.) is then added and the mixture filtered through a filter-press. The filter-cake is Washed With water (50 litres). The organic phase is separated and washed with Water (11 litres) at pH 9, and then with water (11 litres) at pH 5. The organic layer is again separated and concentrated under reduced pressure to of the initial volume of the culture, i.e. 1.9 litres. After standing for 24 hours in a cold room, the precipitate obtained is separated, washed and dried, giving a crude product (88 g.) having an activity of 434 ,ug./mg.

EXAMPLE III The product obtained in Example II (70 g.), titre 434 ng/mg, is suspended in methanol (1400 cc.) and agitated for 1 hour at 30 C. The insoluble matter is separated and dried, giving a product (29 g.) having an activity of 825 ,ug/mg. This product is dissolved in a chloroformme'thanol mixture (90: 10 by volume; 750 cc.) The solution is filtered and then chromatographed through a column containing alumina (600 g.; height of column 61 cm., diameter 3.5 cm.). The chromatogram is developed and eluted with the same chloroform-methanol mixture (1500 cc.). The rich fractions are combined, concentrated under reduced pressure to 40 cc. and precipitated with hexane (400 cc.). The precipitate is separated, washed and dried, giving a product (23 g.) having an activity of 950 ,ug/ mg. A suspension of the latter product (10 g.) in glacial acetic acid (200 cc.) is heated to 50 C. for 5 minutes. The solution is filtered, water added (2.5 cc.) and cooled slowly. The crystals obtained are separated, washed .and dried, giving a pure product (8 g.) having an activity of 1000 .tg./ mg.

EXAMPLE IV A fermentation culture litres) having an activity of 166 ug/cc. is placed in a container fitted with an agitator and adjusted to pH 5 with dilute sulphuric acid. Ethyl acetate (95 litres) is added and the mixture agitated for 1 hour. The organic phase is separated by centrifuging in a centrifuge capable of separating an organic solvent from an aqueous suspension of insoluble material (Westphalia SKOG 205). The organic solution (82 litres) which is recovered is washed and then concentrated under reduced pressure to 1.7 liters. After treatment as described in Example H, a product (61 g.) having an activity of 320 ,ug./ mg. is obtained. This product (35 g.) is suspended in methanol (700 cc.) and agitated for 1 hour at 30 C. The

precipitate is separated and dried, giving a product (11.1 i g.) having an activity of 725 ,ug/mg. By dissolving this product (10 g.) in glacial acetic acid (200 cc.) and treating as described in Example Ill, a crystalline product (6.3 g.) is obtained having an activity of 925 ,ug./ mg.

We claim:

1. An antibiotic, herein designated 9671 RP, which is a yellow substance, crystallising as fine needles, melting point 310320 C. with decomposition on a Maquenne block, and having an optical rotation [a] =+38 (6:1, pyridine), analysing with the elementary composition C, 49.6%; H, 4.0%; O, 16.7%; N, 14.4%; S, 15.75- 15.80%, having'an ultra-violet spectrum in solution in water containing dirnethylformamide exhibiting two absorption maximaat exhibiting the following principal infrared absorption bands 3350 strong, 3125 shoulder, 2930 shoulder, 1745 medium, 1655 very strong, 1532 very strong, 1484 strong, 1425 medium, 1385 weak, 1342 medium, 1308 medium, 1235 medium, 1210 medium, 1168 medium, 1150 medium, 1112 shoulder, 1101 medium, 1061 medium, 1017 medium, 990 weak, 940 weak, 918 medium, 890 shoulder, 843 medium, 822 weak, 790 medium and 752 strong, the said antibiotic being soluble in chloroform, dioxane, pyridine, dimethylformamide and dimethylsulphoxide, slightly soluble in methanol, ethanol, ethyl acetate and benzene and insoluble in water and petroleum ether.

2. A method for the production of an antibiotic, which comprises culturing aerobically the micro-organism S. actuosus, identified by Culture Collection number NRLL 2954 at 23-35 C., on a nutrient medium containing assimilable sources of carbon and nitrogen and having an ini- -tial pH of 6.0 to 7.5 and separating from the culture by ex traction with an organic solvent an antibiotic which is a yellow substance, crystallising as fine needles, melting point 310320 C. with decomposition on a Maquenne block, and having an optical rotation [a] =+38 (c: 1, pyridine), analysing with the elementary composition C, 49.6%; H, 4.0%; O, 16.7%; N, 14.4%; S, 15.75-15.80%, having an ultra-violet spectrum in solution in water containing dimethylformamide exhibiting two absorption maxima at exhibiting the following principal infra-red absorption bands 3350 strong, 3125 shoulder, 2930 shoulder, 1745 medium, 1655 very strong, 1532 very strong, 1484 strong, 1425 medium, 1385 weak, 1342 medium, 1308 medium, 1235 medium, 1210 medium, 1168 medium, 1150 medium, 1112 shoulder, 1101 medium, 1061 medium, 1017 medium, 990 week, 940 weak, 918 medium, 890 shoulder, 843 medium, 822 weak, 790 medium and 752 strong, the said antibiotic being soluble in chloroform, dioxane, pyridine, dimethylformamide and dimethylsulphoxide, slightly soluble in methanol, ethanol, ethyl acetate and benzene, and insoluble in water and petroleum ether.

3. A method according to claim 2, wherein the culturing is effected by the submerged aerobic culture technique with an aeration rate of 0.5 to 2 litres of air per litre of nutrient medium per minute for a period of 3 to 5 days.

4. A method according to claim 2, wherein the temperature of the culture is maintained at 26-27 C.

5. A method according to claim 2 wherein the separation is effected at a pH of 1 to 6.

6. A method according to claim 2, wherein the antibiotic separated from the culture medium is purified by crystallisation from solution in acetic acid.

References Cited in the file of this patent J. Antibiotics (Tokyo), Ser. A 14, 196, 255 1961). J. Am. Chem. Soc. 82, 4747 (1960); 83, 3906 (1961). 

1. AN ANTIBIOTIC, HEREIN DESIGNATED 9671 RP, WHICH IS A YELLOW SUBSTANCE, CRYSTALLISING AS FINE NEEDLES, MELTING POINT 310-320* C. WITH DECOMPOSITION ON A MAQUENNE BLOCK, AND HAVING AN OPTICAL ROTATION (A)D20=+38* (C=1, PYRIDINE), ANALYSING WITH THE ELEMENTARY COMPOSITION C, 49.6%; H, 4.0%; O, 16.7%; N, 14.4%; S, 15.7515.80%, HAVING AN ULTRA-VIOLET SPECTRUM IN SOLUTION IN WATER CONTAINING DIMETHYLFORMAMIDE EXHIBITING TWO ABSORPTION MAXIMA AT 242 M$ (E = 525) AND 322 M$ (E = 229) EXHIBITING THE FOLLOWING PRINCIPAL INFRA-RED ABSORPTION BANDS 3350 STRONG, 3125 SHOULDER, 2930 SHOULDER, 1745 MEDIUM, 1655 VERY STRONG, 1532 VERY STRONG, 1484 STRONG, 1425 MEDIUM, 1385 WEAK, 1342 MEDIUM, 1308 MEDIUM, 1235 MEDIUM, 1210 MEDIUM, 1168 MEDIUM, 1150 MEDIUM DIUM, 990 WEAK, 940 WEAK, 918 MEDIUM, 890 SHOULDER 843 MEDIUM, 822 WEAK, 790 MEDIUM AND 752 STRONG, THE SAID ANTIBIOTIC BEING SOLUBLE IN CHLOROFORM, DIOXANE, PRYIDINE, DIMETHYLFORMAMIDE AND DIMETHYLSULPHOXIDE, SLIGHTLY SOLUBLE IN METHANOL, ETHANOL, ETHYL ACETATE AND BENZENE AND INSOLUBLE IN WATER AND PETROLEUM ETHER. 